The Crest Phenotype in Chicken Is Associated withEctopic Expression of HOXC8 in Cranial SkinYanqiang Wang1, Yu Gao1, Freyja Imsland2, Xiaorong Gu1, Chungang Feng1, Ranran Liu1, Chi Song1,3,

Michele Tixier-Boichard4, David Gourichon5, Qingyuan Li1, Kuanwei Chen3, Huifang Li3,

Leif Andersson2,6, Xiaoxiang Hu1*, Ning Li1*

1 State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, China, 2 Department of Medical Biochemistry and Microbiology, Uppsala University,

Uppsala, Sweden, 3 Jiangsu lnstitute of Poultry Science, Yangzhou, China, 4 INRA, AgroParisTech, UMR1313 Animal Genetics and Integrative Biology, Jouy-en-Josas,

France, 5 INRA, UE1295 PEAT, Nouzilly, France, 6 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden

Abstract

The Crest phenotype is characterised by a tuft of elongated feathers atop the head. A similar phenotype is also seen inseveral wild bird species. Crest shows an autosomal incompletely dominant mode of inheritance and is associated withcerebral hernia. Here we show, using linkage analysis and genome-wide association, that Crest is located on the E22C19W28linkage group and that it shows complete association to the HOXC-cluster on this chromosome. Expression analysis oftissues from Crested and non-crested chickens, representing 26 different breeds, revealed that HOXC8, but not HOXC12 orHOXC13, showed ectopic expression in cranial skin during embryonic development. We propose that Crest is caused by acis-acting regulatory mutation underlying the ectopic expression of HOXC8. However, the identification of the causativemutation(s) has to await until a method becomes available for assembling this chromosomal region. Crest is unfortunatelylocated in a genomic region that has so far defied all attempts to establish a contiguous sequence.

Citation: Wang Y, Gao Y, Imsland F, Gu X, Feng C, et al. (2012) The Crest Phenotype in Chicken Is Associated with Ectopic Expression of HOXC8 in CranialSkin. PLoS ONE 7(4): e34012. doi:10.1371/journal.pone.0034012

Editor: Yi Li, Central China Normal University, China

Received October 12, 2011; Accepted February 20, 2012; Published April 13, 2012

Copyright: � 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by the National Advanced Technology Research and Development Program of China (No. 2011AA100301), National NaturalScience Foundation of China (Grant No. U0831003), grants from the State Key Laboratory of Agrobiotechnology (2011SKLAB02-3) and the Program for NewCentury Excellent Talents in University (NCET-08-0527). The funders had no role in study design, data collection and analysis, decision to publish, or preparation ofthe manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: huxx@cau.edu.cn; ninglcau@cau.edu.cn

Introduction

A feather-crested head is a prominent feature exhibited by

several wild bird species, as well as varieties of several

domesticated birds [1]. In chickens Crest (Cr) is an autosomal

incompletely dominant mutation that causes a tuft of elongated

feathers to sprout from the head, with homozygous individuals

often exhibiting a more developed crest than heterozygotes. The

phenotype shows a degree of sexual dimorphism, with males

exhibiting more voluminous crests than females. Homozygosity for

Crest has been associated with cerebral hernia that causes a

malformation of the cranium [2,3,4,5,6].

The earliest known description of chickens with a Crest comes

from the Roman author Claudius Aelianus, around the turn of the

3rd century AD. Archeological evidence indicates that crested

chickens may have been among the earlier differentiated domestic

varieties, having been discovered in a Roman era site in Britain

that is presumed to have been active in the 4th century AD [7].

Scientific studies of the Crest phenotype go back more than 100

years. In 1906, a number of crosses involving the crested varieties

Polish, Houdan and Silkie were set up by Davenport [8]. In 1928,

Serebrovsky and Petrov published the first genetic linkage map of

any domestic animal, including eight linkage groups in chickens,

and the Crest locus was included in this map [9]. Further breeding

experiments by various scientists have established Crest as

belonging to linkage group II, which today is known to also

contain the classical phenotypic loci Fray (Fr), Dominant white (I) and

Frizzle (F) [10,11,12,13] In 1934, Fisher reported that Crest was

inherited in Mendelian proportions and was dominant over

crestless head [5]. In 2004, the Dominant white locus was mapped to

E22C19W28 linkage group (LGE22C19W28) [14]. This placed

linkage group II, including Crest in the molecular LGE22C19W28

linkage group.

The chicken genome is composed of 38 pairs of autosomes and

a pair of sex chromosomes. The autosomes are of vastly different

sizes and are classified as either macro-chromosomes or micro-

chromosomes. LGE22C19W28 has not yet been assigned to a

chromosome, but it is expected to reside on a micro-chromosome.

For some micro-chromosomes and linkage groups, the chicken

genome is still relatively deficient in markers. In the chicken

consensus linkage map the length of LGE22C19W28 was estimated

to be 50 cM with five microsatellite markers and the Dominant white

locus [15]. In March 2004 the first draft of the chicken genome

was released, in which the physical map size of LGE22C19W28

was about 70 Kb [16,17]. In the Gallus_gallus-2.1 assembly,

LGE22C19W28 and LGE50C23 were merged as LGE22C19-

W28_E50C23. As the map length of LGE50C23 was 40 cM [15]

the combined map length of LGE22C19W28_E50C23 was then

estimated at 90 cM, containing 900 kb ordered sequences and

190 kb random sequences.

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In this study, we map Crest to the HOXC cluster in the

LGE22C19W28_E50C23 linkage group and report that the Crest

trait is associated with ectopic expression of HOXC8 in the cranial

skin where the Crest develops.

Results

The Crest trait and the characteristics of the cranialfeathers

The Crest phenotype is characterised by a tuft of elongated

feathers atop a chicken’s head (Fig. 1A–H). This trait occurs in

diverse chicken breeds from all over the world including the

Appenzeller, Crevecœur, Icelandic, Jinhuwu, Polish, Silkie,

Sulmtaler, Sultan and Chinese fatty chicken. The appearance of

the Crest depends on the length and shape of the cranial feathers.

Individuals from a single Silkie population from southern China,

segregating for Crest, were sampled for cranial feathers at the age

of 45 weeks. Measurements revealed a significant difference in

feather length between Crested and wild-type chickens (P,0.001)

(Figs. 1I & 1J). The feather shape is also different between roosters

and hens. The Crest of a rooster is composed of feathers that come

to a sharp point whereas the Crest of a hen is composed of

rounded feathers (Figs. 1C & 1D).

Segregation of the Crest phenotype in the CAU F2

resource populationIn this study, we constructed an F2 resource population derived

from reciprocal crosses between the Crested Silkie and the non-

Figure 1. Crested and wild-type chickens. (A–D) Crest phenotype; (E–H) wild-type phenotype; (A and E) Silkie male; (B and F) Silkie female; (C)Chinese fatty chicken male; (D) Chinese fatty chicken female; (G) Chahua chicken male; (H) Chahua chicken female. (I) Overview of cranial featherswith gender indicated, sampled from 44 weeks old Silkie chickens. (a) Crested male; (b) Crested female; (c) Wild-type male; (d) Wild-type female. (J)There was a statistically significant difference in feather length between phenotypes. The number of feathers in each group obtained from threeindividuals were as follows: Crested male: 47; Crested female: 44; wild-type male: 100; wild-type female: 100 (**, P,0.01).doi:10.1371/journal.pone.0034012.g001

Chicken Crest Trait Is Associated with HOXC8 Gene

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crested White Plymouth Rock. All F1 individuals had the Crest

phenotype consistent with a dominant inheritance. Over 3,000 F2

offspring were produced by inter-mating the F1 individuals. The

observed ratio between Crested and non-crested F2 progeny

(2324:789) did not deviate significantly from the expected 3:1 ratio

(x2 = 0.10, d.f. = 1; P = 0.75).The result was fully consistent with

previous studies showing that Crest is controlled by a single

autosomal locus.

Comparative alignment and extending theLGE22C19W28_E50C23 linkage group

Comparative genomic data for chicken and human, based on

DNA-DNA alignments, orthologous protein information and

large-scale synteny data are provided in the Ensembl database

[18]. The data indicate extensive conserved synteny between the

human and chicken genomes. LGE22C19W28_E50C23 of chicken

corresponds to an orthologous region on human chromosome 12

(HSA12) near the 50 Mb region. The region between 45 Mb to

60 Mb on HSA12 shows patches of conserved synteny with other

parts of the chicken genome including chicken chromosomes

GGA1, GGA2, GGA7 and ChrUn_random, the latter consists of

contigs that could not be localised to any specific chromosome.

From position 46.34 Mb to 61.33 Mb on HSA12, there are 80

potential protein-coding genes orthologous between human and

chicken, 38 of these have not been mapped to specific

chromosomes, but are located on 29 contigs. Together these

contigs comprise 739 kb of sequence and include the HOXC gene

cluster which has an essential role in vertebrate development

[19](Table S1).

We tried to extend the LGE22C19W28_E50C23 linkage group

by linkage analysis in the CAU F2 population using eight

microsatellites. Five of these markers were derived from unas-

signed contigs for which the comparative data suggested that they

may be located in LGE22C19W28_E50C23 (Table S2). The

marker SKL0185, belonging to contig15741, was located on

LGE22C19W28 in the Gallus_gallus-1.0 assembly (2004), but was

missing in the Gallus_gallus-2.1 assembly (2006). The marker

SKL0019 is located in the same contig as marker MCW0188,

which has been assigned to LGE22C19W28 [15]. Three additional

markers, SKL0067, SKL0071 and ROS0306 are from unassigned

contigs showing sequence homology to HSA12. Three other

markers. SCN8A [20], MCW0317 and SKL0052 are located on

LGE22C19W28_E50C23, and were selected to anchor and build

the linkage map. The linkage analysis conclusively demonstrated

that all eight markers map to LGE22C19W28_E50C23 (data not

shown).

The markers were further analysed with respect to the Crest

locus using a pedigree material comprising 88 individuals. Crest

showed genetic linkage to all eight microsatellite markers,

SKL0019, MCW0317, SKL0185, SCN8A, SKL0071, SKL0067,

ROS0306 and SKL0052 (Table 1). The linkage analysis revealed

no recombination between SKL0071 and Crest (LOD score = 4.82),

or between Crest and SKL0067 (LOD score = 5.42).

Genome wide association study (GWAS) and finemapping of Crest

278 individuals of the CAU resource population, belonging to

15 full-sib pedigrees, were used for a whole genome association

study and fine mapping analysis. A total of 43,131 SNPs were used

together with the microsatellite marker HOXC8-ssr and the SNP

HOXC8-3end that are located upstream and downstream of

HOXC8, respectively. These two markers were identified by

analysis of a bacterial artificial chromosome (BAC) from the

HOXC cluster region and the polymorphisms were detected by

analysing Crested and wild-type individuals (data not shown).

All the genotyping information was analysed in genome-wide

association study (GWAS) and linkage analysis. We found that

HOXC8-ssr, HOXC8-3end and HOXC11 all showed highly

significant significant linkage to Crest (P = 5.5610233) with no

recombination event detected (Fig. 2, Table S3 and S4). This

suggests that the mutation underlying the Crest phenotype might

be located within the HOXC cluster, in the vicinity of HOXC8.

The HOXC cluster is not properly assembled in the chicken

genome but the human HOXC cluster contains eight HOXC

genes (HOXC4-6 and HOXC8-13) in tandem over a 100 kb region

on HSA12.

In total 117 genetic markers were used to construct a linkage

map of LGE22C19W28_E50C23, 18 of these markers were derived

from unassembled regions that we now mapped to this linkage

group.

Fine mapping of Crest using the INRA resourcepopulation

An INRA resource population segregating for a number of traits

derived from European chicken breeds, including Crest was utilised

as a population of Crested birds distantly related to Chinese

Crested breeds. An F1 cross using two Cr/cr+ males from this

population, crossed with 16 inbred cr+/cr+ White Leghorn females

yielded 383 hatched chicks, 186 Crested, 196 non-crested and one

whose phenotype could not be determined.

A panel of four microsatellites and 11 SNPs were genotyped for

mapping Crest in this population. The markers were selected for

their association with Linkage group II, LGE22C19W28_E50C23

or HOXC8 (Table S5 and S6).

SNPs in the immediate vicinity of HOXC8, one in an intron and

another in the 39 downstream region showed no recombination

with Crest in this material (Table 2).

Expression analysis of typical HOXC genesIn mice, Hoxc12 is reported to have an expression pattern

limited to the epidermal cells of the hair follicle [21]. Overex-

pression of Hoxc13 in the hair follicles of transgenic mice resulted

in baldness accompanied by pathologically dry, flaky and scaly

skin. Interestingly, Hoxc13 knockout mice also showed a phenotype

resulting in baldness [22,23]. Chicken HOXC8 is reported to be

expressed during embryonic development in the dorsal dermal

and epidermal cells during the first stage of feather morphogenesis

[24]. Because mammalian hair and avian feathers are similar

Table 1. Two-point linkage analysis of the Crest locus in theCAU F2 population using eight microsatellite loci on chickenLGE22C19W28_E50C23.

Locus Marker Recombination Fraction LOD Score

Crest SKL0019 0.10 4.50

Crest MCW0317 0.09 5.83

Crest SKL0185 0.11 5.76

Crest SCN8A 0.06 7.48

Crest SKL0067 0 5.42

Crest SKL0071 0 4.82

Crest ROS0306 0.05 8.04

Crest SKL0052 0.06 6.08

doi:10.1371/journal.pone.0034012.t001

Chicken Crest Trait Is Associated with HOXC8 Gene

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structures, both being derivatives of ectoderm, these three Hox

genes (HOXC8, HOXC12 and HOXC13) were chosen as candidate

genes for the Crest locus.

Four embryonic developmental stages were chosen (E8, E10,

E12 and E16) for expression analysis using Crested (Silkie) and

wild-type (White Leghorn) embryos. RT-PCR analysis revealed

that only HOXC8 showed phenotype-specific expression in the

cranial skin of Crested chickens (Fig. 3A). The same expression

difference was confirmed in 26 local Chinese chicken breeds, 4 of

which are fixed for Crest and 22 of which are fixed for wild-type,

as well as in a full-sib Silkie pedigree segregating for Crest

(Figs. 4A&4C, Table S7). Expression profiling was also performed

in other tissues from a Crested and a wild-type Silkie, both at 12

weeks of age. Expression analysis by quantitative PCR (q-PCR)

analysis using different skin tissues from embryonic stage E13 to

E21 showed a trend of increased expression as the embryo

Figure 2. Manhattan plot of genome-wide association analysis for the Crest phenotype in chicken. The x-axis shows the physicalposition of the SNPs by chromosome, and the y-axis shows -log10(p-values). A cutoff value of 7.63 declares a genome-wide highly significantassociation (P,0.001) with a Bonferroni correction. The microsatellite marker HOXC8–ssr that had the most significant p-value is located onLGE22C19W28 by HOXC8.doi:10.1371/journal.pone.0034012.g002

Table 2. Two-point linkage analysis of the Crest locus in the INRA resource population and 15 marker loci.

Locus Marker Recombination fraction LOD score Method Locationa Contig

Crest MCW317 0.09 61.37 Microsatellite E22C19W28_E50C23 Contig633.3

Crest SCN8A 0.08 47.58 Microsatellite E22C19W28_E50C23 Contig164.44

Crest E22-164.33 0.07 69.87 TaqMan E22C19W28_E50C23 Contig164.33

Crest E22-164.30 0.07 70.14 TaqMan E22C19W28_E50C23 Contig164.30

Crest HOXC-SCF2 0.04 18.78 Pyrosequencing Un_random/BAC WAG-73F24 Contig1453.1

Crest HOXC8-INTR 0 112.59 TaqMan WAG-73F24

Crest HOXC8-3end 0 110.18 TaqMan Un_random/BAC WAG-73F24 Contig11389.1

Crest E22-9570 0.02 96.58 TaqMan Un_random Contig9570.1

Crest HOXC-SCF1 0.08 6.54 Pyrosequencing Un_random/BAC WAG-73F24 Contig18292.1

Crest MYG1 0.04 83 TaqMan Un_random/Linkage group II Contig303.3

Crest ROS0306 0.04 69.63 Microsatellite Un_random/Linkage group II Contig303.14

Crest MCW188 0.06 31.17 Microsatellite Linkage group II

Crest E22-663.2 0.23 24.36 TaqMan E22C19W28_E50C23 Contig663.2

Crest E22-186.17 0.24 22.54 TaqMan E22C19W28_E50C23 Contig186.17

Crest E22-186.52 0.25 20.58 TaqMan E22C19W28_E50C23 Contig186.52

The lower LOD scores reported for HOXC-SCF1 and HOXC-SCF2 reflect the fact that only individuals with a recombination event between MCW317 and E22-186.2 weretyped for these markers.aUn_random means markers from unassigned contigs; WAG_73F24 represents markers derived from sequences found in BAC WAG_73F24.doi:10.1371/journal.pone.0034012.t002

Chicken Crest Trait Is Associated with HOXC8 Gene

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Figure 3. Expression analysis of HOXC genes in Silkie (crested) and White Leghorn (non-crested) chickens. (A) HOXC8, HOXC12 andHOXC13 genes were detected by RT-PCR in three tissues (cranial skin, dorsal skin and heart) of Silky and White Leghorn chickens at fourdevelopmental stages (E8, E10, E12, E16). C: Cranial skin; D: Dorsal skin; H: Heart. GAPDH was used as a positive and normalised control. -RT indicates areaction without reverse transcriptase. CK indicates a reaction with water as the template. (B)&(C) q-PCR results for HOXC8 expression level in cranialskin (B); and in dorsal skin (C) during chicken developmental stages E13 to E21 comparing Crested and non-crested chickens. No HOXC8 expressionwas detected in cranial skin from non-crested birds. (*, P,0.05; **, P,0.01).doi:10.1371/journal.pone.0034012.g003

Chicken Crest Trait Is Associated with HOXC8 Gene

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developed (Fig. 3B&C). Apart from the skin tissues, HOXC8

expression was only detected in kidney and muscle, indicating the

gene is not a widely expressed transcription factor in chicken

(Fig. 4B). Northern blot and whole mount in situ hybridisation was

also attempted but we failed to obtain a clear positive signal (data

not shown). Based on the q-PCR results, we suggest that low

expression levels of HOXC8 could explain the poor signal.

To detect downstream targets that might function in a network

with HOXC8 during feather development, we examined the

expression levels of 11 genes, BMP4, BMP2, FGF2, CTNNB1,

WNT11, NOG, WNT3A, SHH, FST, BMP7 and TGFB2 by q-PCR

in cranial skin tissue in 12 weeks old adult Crested and wild-type

Silkies. We found that of these 11 genes, only BMP7 showed a

significant difference in expression between the different genotypes

(P,0.05) (Fig. 5). It could be speculated that HOXC8 and BMP7

might act together in the same pathway that affects feather growth.

Loss-of -function and gain-of -function analysis would be necessary

for testing this hypothesis.

Characterisation of chicken HOXC8 by sequencingThe genomic sequence of HOXC8 (Gene ID: 395711) in chicken

was only 405 bp long (NW_001477359) in the Gallus_gallus-2.1

chicken genome release, whereas the chicken HOXC8 mRNA

sequence is 1476 nt (NM_204893). A White Leghorn BAC clone,

WAG-73F24, containing HOXC8 and HOXC13 was identified in a

previous study [25]. Therefore we sequenced this BAC clone,

obtaining 171,765 bp of sequence, from which 86 scaffolds were

assembled. HOXC8 and HOXC6 were located in scaffold 3,

HOXC11, HOXC10, HOXC9 in scaffold 2 and HOXC13 in scaffold

1. We found that the chicken HOXC8 gene is made up of two

exons and one intron (Fig. 6). The result is consistent with the two-

exon structure of human HOXC8 (Gene ID: 3224) and mouse

Hoxc8 (Gene ID: 15426).

Due to the high rate of repetitive and polynucleotide sequence

structures in the HOXC8 genomic region, there are still 20 unfilled

gaps in scaffold 3 after our BAC sequencing effort. This has made

it impossible to completely resequence HOXC8 and its flanking

region with a PCR-based approach in the search for candidate

causative mutations.

Discussion

Crest is associated with ectopic expression of HOXC8This study has provided conclusive evidence that the Crest

mutation is located in the linkage group LGE22C19W28_E50C23 in

the near vicinity of HOXC8. In the CAU F2 population no

recombination was detected between Crest and two genetic

markers, HOXC8-ssr and HOXC8-3end, that are in the immediate

vicinity of HOXC8 for 338 and 221 informative meioses respectively.

The linkage mapping for the INRA population shows the same

association. This result contradicts a previous report based on

fluorescent in situ hybridisation (FISH) that indicated that the

HOXC cluster is located on chicken chromosome 1 and also the

NCBI GenBank description about the position of chicken HOXC8

(Gene ID: 395711, at NW_003778389.1, from 237 bp to 1871 bp)

[26]. We have also demonstrated that the Crest phenotype is

associated with ectopic expression of HOXC8 in cranial skin during

development which appears to be a reasonable explanation for the

atypical feather growth on the heads of Crested chickens. We

therefore propose that Crest is caused by a cis-acting regulatory

mutation that underlies the ectopic expression of HOXC8 that we

have confirmed in four different breeds of Crested chickens. Such a

regulatory mutation may be located in a region corresponding to

one of the HOXC8 regulatory elements that have been defined in

other species, including a microRNA binding site, an early enhancer

element, and a long-range regulatory sequence in the downstream

region [27,28,29]. However, we have not been able to identify the

causative mutation as it has not yet been possible to assemble this

chromosomal region. The LGE22C19W28_E50C23 linkage group

is most likely located on a microchromosome and several of these

have been extremely difficult to assemble due to high GC content

and high density of various forms of repetitive sequences. The

HOXC cluster is not included in the current genome assembly for

chicken. We were not able to assemble it using BAC sequencing and

we have failed to use long-range PCR to connect contigs established

Figure 4. RT-PCR analysis of HOXC8 in different tissues invarious chicken breeds. (A) RT-PCR analysis in skin samples from theprogeny of a heterozygous Cr/cr+ chicken. (B) RT-PCR of seven tissuesfrom Crested and wild-type Silkie chickens. Samples were collected fromtwo individuals at 12 weeks of age. -RT indicates a reaction withoutreverse transcriptase. CK indicates negative control. (C) RT-PCR resultsfor the cranial skin in adult individuals of 26 Chinese local chickenbreeds. Breeds are as follows: 1. Lu Yuan; 2. Gu Shi; 3. Hu Xu; 4.ChineseFatty Chicken; 5. Xian Ju; 6. Da Gu; 7. Silkie; 8. Dou Chicken; 9. Green EggChicken; 10. Wen Chang; 11. You Xi Ma; 12. Ai Jiao Huang; 13. ShouGuang; 14. An Ka; 15. Bian Chicken; 16. White Ear; 17. Kuai Da Silkie; 18.Cha Hua; 19. Yin Xing Bai; 20. Chong Ren Ma; 21. Wa Hui; 22. Jin Hu Wu;23. Tibet Chicken; 24. Shi Qi Za; 25. Black Lang Shan; 26. Qing Yuan Ma;CK: Negative control. Numbers in red indicate chicken breeds with Crestphenotype and black numbers wild-type breeds (Table S12).doi:10.1371/journal.pone.0034012.g004

Chicken Crest Trait Is Associated with HOXC8 Gene

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by BAC sequencing. Therefore, the molecular characterisation of

the Crest causative mutation has to await until a method for

assembling the dark side of the chicken genome (primarily

microchromosomes) has been established.

Chicken HOXC8 and feather developmentHox homeodomain transcription factors play a crucial role in

animal embryonic development. They regulate numerous devel-

opmental pathways and are involved in cellular processes such as

organogenesis, cellular differentiation, cell adhesion and migra-

tion, cell cycle and apoptosis [30]. Morphological diversity

between different groups of animals is under considerable

influence from Hox genes [31,32,33].

Feather development involves a series of interactions between

epithelium and mesenchyme, resulting in the formation of a

specialised keratinous appendage, the feather. The formation of a

Figure 5. RT-PCR analysis of genes involved in feather development in cranial skin from Crested and wild-type chickens (*, P,0.05).GAPDH was used as a normalising control. Relative expression levels were obtained by the 22DDCT method.doi:10.1371/journal.pone.0034012.g005

Chicken Crest Trait Is Associated with HOXC8 Gene

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structure as complex as a feather necessitates a complex pattern of

interactions and signals. Among the signaling molecules that have

essential roles in feather development are bone morphogenetic

proteins 2 and 7 (BMP2 and BMP7), transforming growth factor b2

(TGFB2), b-catenin (CTNNB1), fibroblast growth factors 2 and 4 (FGF2

and FGF4), fibroblast growth factor receptors (FGFRs), noggin(NOG),

Follistatin(FST), WNT3A, WNT7A, WNNT11, sonic hedgehog

(SHH), Delta-1 and epidermal growth factor (EGF) [34,35].

HOX genes are considered to be master transcription factors,

with a function at the top of a genetic hierarchy, exerting great

control over a particular pathway. Direct targets of Hoxc8 in mice

include neural cell adhesion molecule (Ncam), cadherin 11 (Cdh11),

osteopontin (Opn), pigment epithelium-derived factor (Pedf) and

zinc finger protein regulator of apoptosis 1 (Zac1). These five

HOXC8 regulated genes are involved in the Wnt, BMP, and FGF

signalling pathways, all of which have been shown to be involved

in feather morphogenesis [36].

In this study, we found that HOXC8 was the only candidate

gene examined with a strikingly altered expression pattern, which

may directly influence the development of feathers, especially in

terms of the morphology of the cranial feathers and thus cause the

Crest phenotype in chicken.

An exception to the traditional Hox colinearity modelHox genes are present in distantly related groups of animals,

including worms, insects, fish, frogs, birds and mammals, and are

thought to have arisen in a bilateral ancestor to these groups.

Comparison of the composition of Hox gene clusters in various

animals indicates that this ancestor had a cluster of at least eight

Hox-class genes The relative order of the genes in Hox clusters is in

general conserved between species. This in turn tends to dictate a

spatially and/or temporally colinear pattern of expression that

progresses according to the chromosomal position of the genes in

the cluster [37,38].

Colinearity was originally described in Drosophila [38], and has

since been widely observed in animals exhibiting an anterior-to-

posterior axial polarity. Some exceptions to the colinearity of Hox

gene expression have been discovered. Murine Hoxc13 is according

to the model of colinearity a posterior gene, associated with the

hindquarters, but it was found to be expressed in hair follicles all

over the body, including those of the whiskers, as well as in the

tongue. This was the first report of an exception to the traditional

model of colinearity [23,39].

Spatial colinearity has been reported to occur for several Hox

genes expressed in developing chicken skin (HOXB4, HOXA7 and

HOXC8). Other Hox genes, for instance the HOXD cluster, did

not show such colinearity in developing chicken skin [40]. In our

study, the HOXC8 gene showed a tissue-specific expression pattern

in cranial skin in birds carrying the Crest mutation. Considering

that HOXC8 is natively expressed in dorsal skin during the initial

stages of feather follicle formation [24] and that the genomic

location of HOXC8 is medial in the HOXC cluster it could be

argued that the expression of chicken HOXC8 in cranial skin of

Crest mutants is also evidence for an exception to the colinearity

model. Yet we cannot at present exclude the possibility that a

chromosomal rearrangement has occurred on the Crest chromo-

some. If such a rearrangement would eliminate HOXC8

expression from its native expression area, a more severe

phenotype might arise, due to the importance of maintaining

normal Hox gene expression for developmental patterning in

bilateral animals.

That HOXC8 is natively expressed in the dorsal dermis around

embryonic day 8 (E8) [24] is something that may tie in with the

sexual dimorphism in feather shape of the Crest. It is analogous to

the sexual dimorphism of feather shape in the upper tail coverts, or

saddle, of the domestic chicken. Saddle feathers in roosters are

elongate and pointed, not dissimilar to the hackles and Crest

feathers. The same feathers in hens are shorter and rounded

(Figs. 1C & 1D). This gives rise to the suggestion that the ectopic

expression of HOXC8 associated with Crest may reprogram the

cranial dermis to act as if it were dorsal dermis.

Materials and Methods

Ethics statementAll the chickens at INRA experimental farm were fed and

sacrificed according to national regulations and standards of

animal welfare. The farm is registered by the ministry of

Agriculture with the license number B37-175-1 for animal

experimentation.

All the chickens at China Agricultural University (CAU) were

fed and sacrificed according to local standards of animal welfare

issues. The study was approved by the animal welfare committee

of China Agricultural University with approval number XK257.

CAU Chicken resource population and DNA samplepreparation

The CAU chicken resource population was derived using an F2

design from reciprocal crosses between Silkie and White Plymouth

Rock chickens. The Silkie line was chosen as one of the native lines

Figure 6. Gene structure of chicken HOXC8. The two most significant genetic markers in the GWAS of Crest are indicated.doi:10.1371/journal.pone.0034012.g006

Chicken Crest Trait Is Associated with HOXC8 Gene

PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34012

that had been maintained for generations as a closed population at

CAU. Silkie is homozygous mutant Crest (Cr/Cr) while White

Plymouth Rock chickens are homozygous wild-type (cr+/cr+).

There were 26 males and 160 females in the F0 population

(comprising 58 Silkies and 128 White Plymouth Rock chickens). A

reciprocal cross produced the F1 generation. The F2 generation

was produced by mating 33 F1 males and 165 F1 females, and over

3,000 F2 offspring were hatched. Blood samples were collected

from all F0, F1 and F2 individuals at 12 weeks of age by superficial

venipuncture of a wing vein. DNA was prepared by standard

procedures [41].

Genotyping and primary linkage analysis bymicrosatellite markers

Microsatellites were PCR amplified in a total volume of 15 ml

containing 40 ng genomic DNA, 1.5 mM MgCl2, 50 mM KCl,

10 mM Tris?HCl (pH = 8.3), 0.1% Triton X-100, 200 mM

dNTPs, 0.01% gelatine, 1 U TaqDNA polymerase (Promega

Corporation, Madison, WI, USA) and 8 pmol of each primer, one

of the primers in each pair was fluorescently labelled. Following an

initial incubation at 94uC for 5 min, amplification reactions were

performed for 35 cycles each with denaturing at 94uC for 30 s,

annealing at 60uC for 30 s, and extension at 72uC for 1 min, and a

final elongation step at 72uC for 10 min. The PCR products were

electrophoresed in 6% polyacrylamide gel using an ABI377

sequencer (Applied Biosystems, Foster City, CA, USA). Fragment

sizes were determined by using the GeneScan 3.1 fragment

analysis software (Applied Biosystems) and allele identification

was performed using the Genotyper 2.1 software (Applied

Biosystems).

Genotype data from four full-sib families, comprising 88 birds,

were analysed for eight microsatellites and the Crest locus (primers

are listed in Table S2). Linkage analysis was performed using the

Crimap software [42]. Initially, the Two-point option of CRI-

MAP was used and the markers were grouped by two-point value.

Then the order of the different loci was checked using the FLIPS

option.

Sequencing of a BAC clone containing HOXC genesDe novo sequencing data for the White Leghorn BAC clone

WAG-73F24 were obtained by sub-cloning and sequencing with

Illumina genome analyser (Illumina, San Diego, USA) in one lane

[25]. All primary sequence data were assembled by the BGI

bioinformatics service (Shenzhen, China). All assembled data for

the BAC clone have been submitted to GenBank with accession

number JN129278.

Genotyping the markers used in GWAS and fine mappinganalysis

A genotyping experiment using Illumina 60 K chicken SNP

Beadchip was outsourced to the Illumina-certified service

provider, DNA LandMarks Inc., Canada. Quality control was

performed in GenomeStudio. One sample was excluded due to a

low call rate (,95%). 14,997 SNPs were removed with the

following metrics: low call frequency (,95%), low heterozygosity

intensity and cluster separation value (,0.4), heritability or

replication error, and low minor allele frequency (,0.1). A total

of 42,639 SNPs were used in the subsequent analysis.

In addition, seven SNPs (rs14741264, rs14689665, rs16019364,

rs16687544, rs16687551, rs16710870 and rs16714644) were

analysed using SNPlex assays (Applied Biosystems, Foster city,

USA). rs14741264 is located in HOXC11 (GeneBank ID:430698)

and it was also aligned to our de novo BAC sequence.

The HOXC8-3end SNP was genotyped using pyrosequencing

(Biotage, Sweden). Primers were designed using the Assay Design

software. The microsatellite marker HOXC8-ssr was amplified

using HotStarTaqPlus DNA Polymerase kit (Qiagen, Cat.

no. 203603, Germany). The PCR products were denatured for

5 min at 94uC before electrophoresis in POP-7TM polymer

(Applied Biosystems) on an ABI3130xl sequencer (Applied

Biosystems). The results were analysed with ABI Genemapper

4.1 software. There were two alleles (314 bp, 322 bp) according to

the length of PCR products.

Genome-wide association study and linkage analysis forLGE22C19W28

Statistical tests for GWAS were carried out using the epiSNP2

computer package (version 3.4), which implemented the extended

Kempthorne model that allows linkage disequilibrium between

SNPs and Hardy-Weinberg disequilibrium of each SNP [43], and

used a two-step generalised least squares analysis that detected

association accounting for sample relationship [44]. To eliminate

some spurious associations, a P-value threshold of 2.361028 was

considered to be genome-wide significance (P,0.001) with a

Bonferroni correction.

Fine mapping for linkage analysis was conducted with the same

markers as used in the GWAS. The linkage map for the

LGE22C19W28 linkage group was constructed on a 64-bit Linux

system with Crimap software (version 2.503) which was kindly

provided by Dr. Jill Maddox. Markers with inheritance errors or

double recombinant error caused by genotyping were checked and

excluded manually. All unassigned SNPs were mapped to

LGE22C19W28 using Crimap ‘‘two-point’’ option, these SNPs

were added to the new linkage group if they showed significant

linkage with markers from LGE22C19W28 (LODscore.3). The

orders of all markers in the new LGE22C19W28 linkage map were

corrected by ‘‘all’’ and ‘‘flips’’ options.

Tissue collection, RT-PCR and quantitative real-time RT-PCR

Tissues samples (heart, liver, spleen, lung, kidney, heart, brain,

muscle, cranial skin and dorsal skin) collected from chicken

embryos or adult chickens were immediately stored in liquid

nitrogen. Total RNA was extracted using Trizol (Tiangen, Beijing,

China) or RNA RNeasy Mini Kit (Qiagen, Cat.no. 74104). The

RNA preparations were treated with RNase free DNase I to

remove potentially contaminating DNA. First-strand cDNA was

synthesised using 2 mg RNA with an oligo-dT primer. RT-PCR

Primers were designed using the Primer3 web tool (http://frodo.

wi.mit.edu/primer3/).

Samples were run in triplicate on ABI Prism 7900 HT

Sequence Detection System (Applied Biosystems) according to

the manufacturer’s protocol. Four individuals of each embryonic

stage were used for each breed in q-PCR experiments to detect the

expression level of HOXC8. GAPDH was used to normalise each

sample. Primers (Table S7) were designed using the Primer

Express Software (Applied Biosystems). q-PCR for HOXC8 from

E13 to E21 was conducted by Taqman Universal PCR Master

Mix (Applied Biosystems) using fluorescent marked probe at the 59

end of the probe. Separately, plasmids containing HOXC8 and

GAPDH gene fragments were diluted to a concentration of 1028,

1027, 1026, 1025 or 1024 to make standard curve. Each 15 ml q-

PCR reaction was made up of 7.5 ml master mix, 150 nM of each

primer, 75 nM of each probe and 1 ml cDNA as template.

q-PCR of eleven genes (BMP4, FGF2, BMP2, CTNNB1, BMP7,

WNT11, NOG, WNT3A, SHH, FST andTGFB2) for pathway

Chicken Crest Trait Is Associated with HOXC8 Gene

PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34012

analysis was conducted in triplicate by the 22DDCT method [45].

GAPDH was used as a normalisation control. Eight individuals

(four for each phenotype, Crest and wild-type) were used. Each

15 ml q-PCR reaction was made up of 7.5 ml master mix, 150 nM

of each primer and 1 ul cDNA as template.

Statistical analysis was performed using SPSS software (version

17.0) for a two-sample t-test on the average of each of the

triplicates.

INRA population and linkage analysis thereofThe resource population at INRA is bred from numerous

European chicken breeds and segregates for various traits found in

European fowl populations. Two roosters from this population,

both heterozygous for Crest were mated to inbred non-crested

White Leghorn hens, eight females for each rooster, 16 hens in

total. Approximately 200 chicks sired by each rooster hatched.

Chicks were produced in two batches hatched three weeks apart.

Phenotypes for Crest were scored by visual examination of each

bird at the age of 46 or 47 days, depending on the batch. Blood

sample for DNA extraction was collected from the wing vein of

each chicken on the day of phenotypic recording.

Linkage analysis for Crest in this population was performed by

genotyping a panel of microsatellite markers and SNPs via

Pyrosequencing and TaqMan genotyping assays. 59-fluorescent

M13 primers combined with a primer with an M13 tail were used

in amplification of microsatellite markers. The PCR products were

denatured for 2 min before electrophoresis in 4% polyacrylamide

gels using a MegaBACE capillary instrument (Amersham Bio-

sciences, Uppsala, Sweden). The results were analysed with

Genetic Profiler software (Amersham Biosciences). Pyrosequenc-

ing was performed using the SNP Reagent Kit protocol

(Pyrosequencing AB, Uppsala, Sweden). 59-biotinylated M13

primers combined with a primer with an M13 tail were used to

allow capture of single stranded products onto avidin-coated

beads. Custom Taqman@ SNP Genotyping Assays (Applied

Biosystems) were used for nine SNPs, run according to

manufacturer’s instructions on a 7900HT Fast Real-Time PCR

system (Applied Biosystem). Genotyping calls were made with SDS

2.3 software (Applied Biosystems). Primers were designed with the

Primer3Plus webtool (http://www.bioinformatics.nl/cgi-bin/

primer3plus/primer3plus.cgi); see Table S5,S6, S8, S9, S10 and

S11 for further method information. Linkage analysis for the

INRA population was performed with Crimap as previously

described for the CAU population.

Supporting Information

Table S1 A list of the potential orthologous genesbetween chicken and human on HSA 12 from 46 Mb to60 Mb.

(DOC)

Table S2 Primers used for genotyping in CAU popula-tion.

(XLS)

Table S3 Two-point linkage analysis for fine mappingof Crest in CAU F2 population and genetic markers onchicken LGE22C19W28.(XLS)

Table S4 Marker alleles associated with the Cresthaplotype derived from Silkie chickens. All markers

showed 1% genome-wise significance.

(XLS)

Table S5 Primers used for analysis of INRA populationmicrosatellites and pyrosequenced SNPs.(XLS)

Table S6 Primers used for amplification and analysis ofINRA population SNPs assayed with a TaqMan Geno-typing Assay.(XLS)

Table S7 Primers used for RT-PCR and q-PCR.(XLS)

Table S8 Amplification conditions used for INRA pop-ulation microsatellites. M13 primers (59-CACGACGTTG-

TAAAACGAC-39) were variously labeled with the fluoresent dyes

Hex, Fam and Tet.

(XLS)

Table S9 PCR conditions for INRA population micro-satellite analysis.(XLS)

Table S10 Amplification conditions used for INRApopulation microsatellites. M13 primer (59-CAC-

GACGTTGTAAAACGAC-39) was labeled with biotin.

(XLS)

Table S11 PCR conditions for INRA population pyrose-quencing reaction.(XLS)

Table S12 Phenotypic information for 26 differentchicken breeds.(XLS)

Acknowledgments

We thank Mr. Hao Qu, Jie Ma and Chenglong Luo in the Institute of

Animal Science, Guangdong Academy of Agricultural Science for

managing chicken breed populations and providing the photos, and

professor Xuemei Deng in China Agricultural University for constructing

the CAU resource population. We also thank professor Groenen MA and

Richard P. M. A. Crooijmans in Wageningen University who had sent us

the White Leghorn BAC clone, Dr. H.H. Chen and Dr. Susanne Kerje for

providing the microsatellite marker primers and professor Yaofeng Zhao

for his suggestions in experiment design.

Author Contributions

Conceived and designed the experiments: NL XH LA. Performed the

experiments: YW FI YG XG CF RL CS QL. Analyzed the data: XG YW

YG FI. Contributed reagents/materials/analysis tools: MT DG KC HL.

Wrote the paper: YW YG FI LA.

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